Project Summary Our global objective is to develop a molecular understanding of how epithelial stem cells undergo morphogenesis and maintain homeostasis in mammalian skin, and to bring our research to a clinical setting. Our focus is on how, in response to local developmental cues, skin stem cells remodel their cytoskeletal connections with intercellular and cell-substratum junctions in order to generate and maintain tissues. Knowledge of the proteins involved and their physiological roles in coordinating these cytoskeletal and adhesion dynamics are key to understanding how skin epithelia form and are maintained and how cellular organization goes awry in hyperproliferative skin disorders, including cancers. During skin development, a single layer of unspecified epithelial progenitors can both stratify to form the surface barrier and also invaginate to form hair follicles (HFs). As morphogenesis proceeds, resident stem cells are set aside so that in adult skin, self-renewing progenitors maintain and repair the skin's barrier and fuel cyclical bouts of hair regeneration. In prior AR27883 research, we showed that whether normal or malignant, all skin progenitors reside at epithelial-mesenchymal interfaces. Progenitors utilize their underlying basement membrane to polarize and orient their spindle, balance growth and differentiation and migrate. We have shown that in part, WNT-signaling is at the root of these polarized cytoskeletal and adhesion dynamics. However, the epithelium must also sense and respond to cell crowding and mechanical cues. How these signals intersect remain unclear. In the next 5 years, we'll address these critical issues by: (1). Determining the governance and importance of cell density in tissue morphogenesis within the epidermis and HFs. (2). Dissecting the roles of acto-myosin cytoskeletal regulators in tissue morphogenesis. (3). Elucidating how the developing epidermis copes with poorly performing cells for the sake of tissue fitness and how this changes once the stratified skin barrier is established. (4). Elucidating the role(s) of differential WNT- signaling and mechanotransduction regulators within developing hair buds. We'll assess how signaling is sensed in a polarized fashion and how cell density, tension and basement membrane production impacts the decision to invaginate rather than evaginate. (5). Applying the knowledge gained in Aims 1-4 to dissect how and why tissue morphogenesis and maintenance change in malignancy. To meet these aims, we'll combine a variety of molecular and genetic approaches. During our R37-AR27883 MERIT Award, we developed live imaging to interrogate cellular movements during skin development. We also pioneered a powerful in utero method to efficiently and selectively compromise gene function in embryonic and adult skin to unveil the physiological relevance of our findings. Finally, we adapted this technology to CRISPR/CAS, enabling us to conduct rapid conditional knockouts in mice as well. Our past and present AR27883 research, displaying continued success over nearly 4 decades, exemplifies how molecular skin biology can lead to new and improved tools for diagnosing and treating human skin diseases. We aim to continue to make such inroads in the next grant period.